In human mitochondria, genes for tRNA(Tyr) and tRNA(Cys) overlap by a single nucleotide. From polycistronic precursors, a 3'-truncated upstream tRNA(Tyr) is released, missing the overlapping position. A subsequent editing reaction restores this position. Similar mitochondrial tRNA gene overlaps exist in all metazoans, but not in organisms such as yeast or Escherichia coli. Therefore, we asked w...

The use of tRNA affinity columns for the purification of aminoacyl-tRNA synthetases was investigated. A purification method for valyl-tRNA synthetase from Bacillus stearothermophilus is described that uses two affinity columns, one containing the pure cognate tRNA, and the other containing all tRNA species except the cognate tRNA. A method for the rapid preparation of the two columns was develo...

The influence of pyrophosphate hydrolysis by inorganic pyrophosphatase on homologous aminoacylation of different yeast tRNA(Phe) mutants was studied. The addition of pyrophosphatase significantly improved the aminoacylation efficiency of tRNA(Phe) structural mutants as well as the mutant with substitution at position 20, while having no effect on the charge of wild-type tRNA(Phe). Aminoacylatio...

Two critical requirements for developing methods for the site-specific incorporation of amino acid analogues into proteins in vivo are (i) a suppressor tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA synthetases (aaRSs) and (ii) an aminoacyl-tRNA synthetase that aminoacylates the suppressor tRNA but no other tRNA in the cell. Here we describe two such aaRS-suppressor tRNA...

There is evidence that tRNA bodies have evolved to reduce differences between aminoacyl-tRNAs in their affinity to EF-Tu. Here, we study the kinetics of incorporation of L-amino acids (AAs) Phe, Ala allyl-glycine (aG), methyl-serine (mS), and biotinyl-lysine (bK) using a tRNA(Ala)-based body (tRNA(AlaB)) with a high affinity for EF-Tu. Results are compared with previous data on the kinetics of ...

Amplification and sequencing of mitochondrial DNA regions corresponding to three major clusters of transfer RNA genes from a variety of species representing major groups of birds and reptiles revealed some new variations in tRNA gene organization. First, a gene rearrangement from tRNA(His)-tRNA(Ser)(AGY)-tRNA(Leu)(CUN) to tRNA(Ser)(AGY)-tRNA(His)tRNA(Leu)(CUN) occurs in all three crocodilians e...

Pyrrolysine (Pyl), the 22nd naturally encoded amino acid, gets acylated to its distinctive UAG suppressor tRNA(Pyl) by the cognate pyrrolysyl-tRNA synthetase (PylRS). Here we determine the RNA elements required for recognition and aminoacylation of tRNA(Pyl) in vivo by using the Pyl analog N-epsilon-cyclopentyloxycarbonyl-l-lysine. Forty-two Methanosarcina barkeri tRNA(Pyl) variants were tested...

Protein synthesis in eukaryotic organelles such as mitochondria and chloroplasts is widely believed to require a formylated initiator methionyl tRNA (fMet-tRNA(fMet)) for initiation. Here we show that initiation of protein synthesis in yeast mitochondria can occur without formylation of the initiator methionyl-tRNA (Met-tRNA(fMet)). The formylation reaction is catalyzed by methionyl-tRNA formyl...

Growing cultures of mutant Escherichia coli with temperature-senstive peptidyl-tRNA hydrolase were shifted to nonpermissive 4o degrees. There followed a roughly linear increase in a fraction of isolated tRNA (over 50% after 20 min) whose amino acid-accepting activity was masked until treatment with active peptidyl-tRNA hydrolase. The ionophoretic mobility of amino acid label associated with thi...

The study of tRNA molecules altered by a mutational event can lead to information in 3 important areas: (a) structure-function relationships in tRNA biochemistry; (ft) the genetics of tRNA; and (c) the physiological role of tRNA-isoaccepting species. For example, the elucidation of the structural alteration in a mutant tRNA species which reacts appreciably more slowly than normal tRNA in the en...